IOCAS-IR
白斑综合征病毒及对虾dUTPase与蛋白类抑制剂Stl相互作用的结构基础
王方
学位类型博士
导师马庆军
2021-07-03
学位授予单位中国科学院大学
学位授予地点中国科学院海洋研究所
学位专业海洋生物学
关键词白斑综合征病毒 凡纳滨对虾 dUTP焦磷酸酶 蛋白类抑制剂 空间位阻
摘要

白斑综合征病毒(White spot syndrome virus,WSSV)可以引发白斑综合征,是虾类乃至整个甲壳类的重要病原。目前针对白斑综合征尚无有效的治疗措施,因此开发抗WSSV病毒药物十分必要。利用病毒复制的关键蛋白为靶标开发特异性抑制剂是防治病毒类疾病普遍采用的策略之一。WSSV是一种大型DNA病毒,其基因组编码了包括脱氧尿苷焦磷酸酶(dUTPase)等的核酸代谢及DNA复制相关的重要酶类。dUTPase可以催化dUTP水解成dUMP和焦磷酸PPi,在胸苷酸代谢中发挥着关键作用,是较为成熟的药物靶标。以WSSV的dUTPase(wDUT)为靶点设计抑制剂有望成为防控WSSV病毒的有效手段。

尽管宿主凡纳滨对虾(Litopenaeus vannamei)dUTPase (lvDUT)及病毒wDUT在结构上存在一些差异,但他们在活性位点的氨基酸组成及催化机制上保持一致。因此,针对酶的活性位点设计wDUT特异性的小分子抑制剂较为困难。相比之下,大分子抑制剂与酶可结合的区域较广,有望区别活性位点之外的差异而特异性抑制wDUT的活性。金黄色葡萄球菌致病岛抑制因子Stl是对dUTPase具有广谱抑制作用的一种大分子蛋白类抑制剂。这提示我们或可将Stl应用于wDUT的特异性抑制。本研究比较了Stl对宿主lvDUT及病毒wDUT的活性影响,并解析了lvDUT-StlN-ter及wDUT-StlN-ter的复合物晶体结构,揭示了Stl与两种dUTPases结合模式的显著差异,为合理的工程改造Stl,开发wDUT特异性的蛋白类抑制剂奠定了坚实的基础。

首先,本课题研究了Stl与凡纳滨对虾lvDUT的相互作用。研究发现Stl不仅可以通过其N端结构域(StlN-ter)与lvDUT结合,还可以抑制lvDUT的活性,且最高的抑制效率为65%左右。解析StlN-ter与lvDUT复合物的结构显示Stl的α8及其周围区域结合在lvDUT的活性中心,占据底物结合位置,形成了复合物的主要相互作用界面。另外Stl的 α5和α7可以通过疏水相互作用结合lvDUT的C末端,防止C末端加入到活性位点组装。通过这两部分相互作用,Stl发挥抑制lvDUT的作用。另外,通过将lvDUT-StlN-ter与以往的Φ11DUT-StlN-ter复合物进行对比,我们推测了Stl对不同物种dUTPase抑制效率产生差异的一些原因。总之,作为Stl与真核dUTPase的第一个复合物结构,lvDUT-StlN-ter复合物结构的解析完善了Stl对三聚体dUTPase的抑制机制,并为真核dUTPases以及针对wDUT的特异性蛋白类抑制剂的研制提供了坚实的基础。

其次,我们探究了Stl与WSSV病毒wDUT的相互作用模式。实验结果显示,Stl与wDUT的结合能力较弱,且对wDUT的活性无显著影响。解析StlN-ter与 wDUT复合物结构发现,Stl与wDUT的结合方式与lvDUT-StlN-ter完全不同。Stl的α2−3及之间的loop上的部分氨基酸主要与wDUT β1与β2之间的loop结合。这种结合方式形成的相互作用界面较小,也是导致Stl与wDUT亲和力较弱的原因。在这种结合模式下,Stl远离了wDUT的活性中心,且对wDUT的整体结构及活性位点结构没有显著的影响,这与Stl不能抑制wDUT活性结果一致。结构模拟分析显示,Stl的α4螺旋与wDUT特有的pre-V区域形成的空间位阻是Stl不能结合在wDUT活性位点的原因。通过截断突变消除空间位阻后Stl与wDUT的结合方式会发生转变。我们将与lvDUT活性中心结合的Stl-N2(87−153)进行关键氨基酸Y112和Y113突变。与结合lvDUT情况一致的是,野生型Stl-N2可以结合wDUT,而关键氨基酸Y112, Y113突变后,其与wDUT的结合能力丧失。这说明改造Stl后,Stl的截短体以类似结合lvDUT的方式结合wDUT。

总之,本研究通过解析两种复合物结构发现Stl与凡纳滨对虾及WSSV的dUTPase结合模式存在差异。这种差异是由于在与wDUT活性位点结合时,Stl的α4与wDUT特有的pre-Ⅴ区域存在空间位阻导致的。本研究在结构的基础上合理的截短Stl,改变了其与wDUT的结合方式,并为进一步优化Stl成为抑制wDUT的特异性蛋白类抑制剂提供了良好的基础。

其他摘要

White spot syndrome virus (WSSV) can cause white spot syndrome and is an important pathogen of shrimp and even the whole crustacean. At present, there are no effective prevention and treatment measures for white spot syndrome, so it is very necessary to develop anti-WSSV drugs. Developing specific inhibitors targeting key proteins of viral replication is one of the strategies widely used in the prevention and treatment of viral diseases. WSSV is a large DNA virus whose genome encodes important enzymes related to nucleic acid metabolism and DNA replication, including dUTPase. dUTPase catalyzes the hydrolysis of dUTP into dUMP and PPi, which is essential in thymidylate metabolism and considered as mature target to design drugs. Design of inhibitors targeting the dUTPase of WSSV (wDUT) is expected to be an effective means of anti-WSSV therapy.

Although there are some structural differences between host Litopenaeus vannamei dUTPase (lvDUT) and virus wDUT, they are consistent in the amino acid composition of the active site and the catalytic mechanism. Therefore, it is difficult to design species-specific small molecule inhibitors targeting the active center of wDUT. In contrast, macromolecular drugs can bind to a wide range of enzymes and are expected to specifically inhibit the activity of wDUT according to the differences beyond the active site. Stl, the repressor of Staphylococcus aureus pathogenicity islands (SaPIs), is a macromolecular proteinaceous inhibitor with broad-spectrum inhibition of dUTPases. This suggests that Stl may be applied to the specific inhibition of wDUT. In this study, the effects of Stl on the enzymatic activity of host lvDUT and virus wDUT were compared, and the crystal structures of the complexes of lvDUT-StlN-ter and wDUT-StlN-ter were resolved, which revealed the significant differences in the binding modes between Stl and the two dUTPases, laying a solid foundation for the rational engineering modification of Stl and the development of wDUT specific proteinaceous drugs.

Firstly, the interaction between Stl and lvDUT has been studied. It was found that Stl could not only bind to lvDUT through its N-terminal domain, but also inhibit the activity of lvDUT, with a maximal inhibition rate of 65%. Crystal structure of the complex between the N-terminal domain of Stl and lvDUT shows that the helix α8 and its surrounding area of Stl were inserted into the active center of lvDUT and occupied the substrate binding position, forming the major interaction interface. In addition, the helix α5 and α7 of Stl can form hydrophobic interaction with the C-terminal of the lvDUT, which hinders the C-terminal tail of lvDUT from adopting the active conformation. Through these interactions, Stl could lead to the enzymatic activity loss of lvDUT. In addition, superposing lvDUT-StlN-ter and previous Φ11DUT-StlN-ter, we speculated some reason for the different inhibitory efficiency of Stl on different dUTPases. Altogether, as the first structural model of Stl interaction with eukaryotic dUTPase, lvDUT-StlN-ter contributes to a more complete view of the inhibition mechanism of Stl on trimeric dUTPases, and facilitates the development of specific proteinaceous inhibitors for eukaryotic dUTPases and wDUT.

Secondly, we investigated the interaction pattern between Stl and wDUT of WSSV. The experimental results show that the binding ability of Stl to wDUT was weak, and it had no significant effect on the activity of wDUT. The crystal structure of wDUT-StlN-ter complex shows a completely different binding pattern from that of lvDUT-StlN-ter complex. The helix α2-α3 and the connecting loop of Stl mainly interact with wDUT β1−β2 connecting loop. The interface formed by this interaction pattern is smaller, which may be the reason for the weak affinity between Stl and wDUT. In this interaction pattern, Stl is far away from the active center of wDUT and has no significant influence on the overall structure and active site of wDUT, which is consistent with the result that Stl could not inhibit the enzyme activity of wDUT. Structural simulation show that the steric hindrance formed by the α4 of Stl and the specific pre-V region of wDUT is the reason why Stl can not bind to the activity center of wDUT. Eliminating steric hindrance by truncation mutation, the binding mode of Stl and wDUT can be changed. We mutated the key residues Y112 and Y113 in the Stl-N2 (87−153), which is responsible for binding to the active center of lvDUT. Consistent with that to lvDUT, Stl-N2 could bind to wDUT, but the binding ability was lost after the mutation of two key residues that bind to the active center of dUTPase. These indicated that after the modification of Stl, the truncation could bind to the active center of wDUT just like binding to lvDUT.

In conclusion, we found that there are differences in binding patterns of Stl to host and WSSV dUTPases by comparing the structures of the two complexes. This difference is attributed to the steric hindrance formed by α4 of Stl and pre-V of wDUT when Stl binding the activity center of wDUT. In this study, we truncated Stl reasonably on the basis of structure, which changed the binding mode between Stl and wDUT, and provided a good foundation for further optimization of Stl as a proteinaceous drug that specifically inhibits wDUT.

学科门类海洋科学
页数135
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/175800
专题中国科学院海洋研究所
实验海洋生物学重点实验室
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GB/T 7714
王方. 白斑综合征病毒及对虾dUTPase与蛋白类抑制剂Stl相互作用的结构基础[D]. 中国科学院海洋研究所. 中国科学院大学,2021.
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